CN106908539B - Method for detecting 1-hydroxybenzotriazole in soil and plants - Google Patents
Method for detecting 1-hydroxybenzotriazole in soil and plants Download PDFInfo
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Abstract
the invention discloses a method for detecting 1-hydroxybenzotriazole in soil and plants. The method comprises the following steps: pretreating a sample; accelerating solvent extraction; preparing a standard working solution; and (3) detecting by using a high performance liquid chromatography-mass spectrometer. The invention utilizes an accelerated solvent extraction technology to establish a rapid, high-efficiency and high-recovery extraction method, and after synchronous purification or solid-phase extraction purification, high performance liquid chromatography-mass spectrometry combined detection is utilized to effectively eliminate matrix interference, wherein the recovery rate of 1-hydroxybenzotriazole in a soil sample is 92.6%, the standard deviation is 2.4%, the matrix effect is 90.7%, the detection limit is 0.15ng/g, and the quantification limit is 0.51 ng/g; the recovery rate of 1-hydroxybenzotriazole in the plant sample is 89.8%, the standard deviation is 1.6%, the matrix effect is 90.3%, the detection limit is 0.69ng/g, and the quantification limit is 2.31 ng/g.
Description
Technical Field
The invention belongs to the field of environmental sample detection, and particularly relates to a method for detecting 1-hydroxybenzotriazole in soil and plants.
Background
Benzotriazole compounds (benzotriazole, BTs) are compounds with benzotriazole heterocyclic structures, have excellent stability and corrosion resistance, and can be complexed on the surface of metal to form a protective layer after being contacted with the metal, so that the corrosion of the metal is prevented. Benzotriazole compounds are widely added to industrial products such as refrigerating fluid, hydraulic fluid, antifreeze, detergent, airplane deicing/anti-icing fluid and the like as additives which are widely used. 1-Hydroxybenzotriazole (HOBT) is a common benzotriazole compound, and the basic information is shown in Table 1.
TABLE 1 basic information of HOBT
In recent years, researches show that a plurality of benzotriazole compounds are detected in environmental media such as rivers, lakes, oceans and sediments, and the benzotriazole compounds are greatly remained in the environment and attract attention as new pollutants along with the use of a large amount of benzotriazole compounds. The benzotriazole compounds enter the environment mainly in two ways, namely directly enter the environment along with the use and the abandonment of products; and secondly, after entering a sewage treatment plant through an urban sewage pipe network, the sewage and the sludge enter the environment along with the discharge of the sewage and the sludge. A large number of researches show that the benzotriazole compound has low removal rate in a sewage treatment plant adopting the traditional activated sludge method, and effluent and sludge both contain the benzotriazole compound with higher concentration. In recent years, activities such as sewage irrigation and sludge agriculture are increasing, and benzotriazole compounds may enter farmland soil, be adsorbed and absorbed by crops, and be accumulated and enriched in a food chain, so that the health and survival of human beings are affected finally.
currently, the detection of benzotriazole compounds is mainly focused on environmental water systems such as rivers, lakes and sediments, but the analysis method of the benzotriazole compounds in soil and plants is less concerned. In addition, benzotriazole compounds have been mainly studied on three compounds, namely benzotriazole, 5-methyl-1H-benzotriazole and 5, 6-dimethyl-1H-benzotriazole, and 1-hydroxybenzotriazole has been studied less. Therefore, in order to know the concentration level and accumulation of 1-hydroxybenzotriazole in soil and plants, a high-efficiency and sensitive analysis method needs to be established for detecting trace 1-hydroxybenzotriazole in soil and plants.
Disclosure of Invention
In order to solve the defects and shortcomings of the prior art, the invention aims to provide an extraction, purification and instrumental analysis method of 1-hydroxybenzotriazole, which is used for qualitative and quantitative detection of the 1-hydroxybenzotriazole in soil and plants.
Accelerated solvent extraction is an extraction method which is widely used in recent years, and can be used for extraction of soil, sediment, sludge or biological samples. Compared with extraction methods such as ultrasonic extraction and Soxhlet extraction, the method has the advantages that the operation of the accelerated solvent extraction method is simpler, the automation degree is high, the extraction time is only 30 minutes generally, and the method is suitable for processing samples on a large scale; the solvent usage amount in the extraction process is greatly reduced, the method is more environment-friendly, and the method is an efficient extraction method. The matrix adsorbent is added into the extraction tank, and the extraction process is synchronous in purification, so that the method has a good purification effect on most soil samples and plant samples with low pigment content, saves the experiment time and improves the sensitivity of the method. For samples with high pigment and fat contents such as straws, grains and the like, solid-phase extraction treatment can be carried out after extraction, further purification is carried out, and the matrix effect is reduced.
The high performance liquid chromatography-mass spectrometer adopts a multi-reaction monitoring mode (MRM), has higher selectivity and sensitivity to a target compound, effectively reduces matrix interference, and is suitable for detecting trace target compounds in a complex environment medium.
The purpose of the invention is realized by the following technical scheme:
A method for detecting 1-hydroxybenzotriazole in soil and plants comprises the following steps:
(1) Sample pretreatment
Pretreating the collected soil sample or plant sample;
(2) accelerated solvent extraction
Extracting the sample pretreated in the step (1) by using an accelerated solvent extraction instrument to obtain a sample solution to be detected;
(3) Preparation of standard working solutions
Weighing standard substances of 1-hydroxybenzotriazole and 1 internal standard substance by using an analytical balance, respectively preparing a stock solution and a working solution, and finally preparing a standard working solution with gradient concentration;
(4) High performance liquid chromatography-mass spectrometer detection
measuring the standard working solution of each concentration gradient in the step (3) in a high performance liquid chromatography-mass spectrometer, and establishing a standard working curve by using an internal standard method; injecting the sample solution to be detected in the step (2) into a high performance liquid chromatography-mass spectrometer under the same conditions for determination, calculating the concentration of 1-hydroxybenzotriazole in the solution through a standard working curve, and then calculating the content of the 1-hydroxybenzotriazole in the sample according to the mass of the sample; the concentration of the 1-hydroxybenzotriazole in the sample solution is in the linear range of the standard working curve, and if the measured concentration exceeds the linear range of the standard working curve, the sample amount can be reduced, and then re-extraction and detection are carried out.
In the step (1), the soil sample pretreatment process comprises the following steps: after the soil sample is collected, removing larger impurities such as stones and the like, drying, grinding and sieving for later use; the pretreatment process of the plant sample comprises the following steps: and (3) after collecting the plant sample, removing silt, drying, and uniformly crushing by using a plant crusher for later use.
In the step (1), the drying can be natural air drying or freeze drying, and if freeze drying is adopted, the sample needs to be pre-frozen in a freezer for 24 hours and then put into a freeze dryer for drying. Drying the soil sample, and grinding the soil sample through a 0.90mm sieve; and (4) drying the plant sample and then crushing the dried plant sample by using a plant crusher. After the sample is ground or crushed, the sample should be extracted as soon as possible, if the batch is large and the extraction cannot be carried out in time, the sample should be stored in a refrigerator or a cold storage at 4 ℃ in a sealed manner.
In the step (2), when the sample is a soil sample or a plant sample with low pigment content (such as a part of plant with low pigment content such as underground part including root, pulp, corncob) the extraction process is as follows: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding a certain amount of matrix adsorbent, tapping the extraction tank to flatten the surface of the matrix adsorbent, then adding a pretreated sample, dropwise adding an internal standard solution after flattening, adding quartz sand after the internal standard solution is volatilized, finally pressing the internal standard solution by putting another piece of filter paper, screwing down the extraction tank, and putting the internal standard solution into an accelerated solvent extraction instrument for extraction; after extraction, the extract is subjected to rotary evaporation to be nearly dry, and then 1mL of methanol is used for constant volume; transferring the sample to a sample injection vial by using an organic phase nylon filter membrane to be detected;
When the sample is a sample with high pigment or fat content (such as a sample with high pigment content in the overground part of most plant stems and leaves and the like, and a sample with high fat content in grains such as wheat grains and corn grains), the color of the extract is deep, the direct on-machine detection matrix has serious interference and poor peak shape, the sample can be purified and then detected on-machine, and the extraction and purification processes are as follows: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding a certain amount of matrix adsorbent, tapping the extraction tank to flatten the surface of the matrix adsorbent, then adding a pretreated sample, dropwise adding an internal standard solution after flattening, adding quartz sand after the internal standard solution is volatilized, finally pressing the internal standard solution by putting another piece of filter paper, screwing down the extraction tank, and putting the internal standard solution into an accelerated solvent extraction instrument for extraction; after extraction, the extract is subjected to rotary evaporation to be nearly dry, and then 1mL of methanol is used for constant volume; taking a Florisil solid-phase extraction column (1000mg, 6mL), pre-washing the Florisil solid-phase extraction column with 10mL of dichloromethane and 10mL of methanol in sequence to activate the small column and remove part of impurities, immediately adding a sample to be purified when the liquid level of a solvent in the column drops to be close to the surface of a filler, receiving a drained liquid with a pear-shaped flask, rinsing a sample bottle with 5mL of methanol, adding a rinsing solution into the Florisil solid-phase extraction column, and repeating twice; the pear-shaped flask received all the leachates, rotary evaporated to near dryness, fixed volume with 1mL of methanol, filtered with an organic phase nylon filter membrane, transferred to a sample injection vial, and tested.
in the step (2), the internal standard solution is preferably deuterated thiabendazole (thiabendazole NH D6).
in the step (2), the matrix adsorbent is silica gel and anhydrous sodium sulfate, and the function of the adsorbent is to synchronously purify in the extraction process, so that the experimental time is saved. Wherein the silica gel is 80-100 meshes, washed by methanol and dichloromethane sequentially for 2 times, dried in the air, and baked in an oven at 120 ℃ for 4 hours for later use. The specific operation of adding the matrix adsorbent is that 1-3 g of silica gel is added firstly, after the surface is flattened, 1-3 g of anhydrous sodium sulfate is added, and the usage amount of the matrix adsorbent is properly increased or decreased according to the sample amount.
in the step (2), the sample amount is preferably 2-8 g. If the interference of the sample matrix is large or the concentration of the target compound is high, the sample amount can be reduced, and the sample and quartz sand with the mass of 1-4 times are uniformly mixed and then added into an extraction pool. After the fluffy samples such as plant straws and the like are added into the extraction tank, the samples can be slightly pressed, and the volume is reduced.
In the step (2), the quartz sand is added as a filling agent, the addition amount is 2-5 g, and the filling agent is determined according to the sample amount, so that on one hand, the space of an extraction pool is filled, and the usage amount of an extraction solvent is reduced on the premise of sufficient extraction; and on the other hand, the sample in the extraction cell is protected, and the matrix adsorbent and the sample are prevented from being dispersed by the solvent injected under high pressure in the extraction process.
In step (2), the preferred extraction conditions are as follows: the capacity of the extraction pool is 34mL, the extraction solvent is a mixed solvent of methanol and dichloromethane, wherein the proportion of the methanol is 80-100% (volume ratio), and in order to improve the recovery rate, formic acid with the total volume of 0.005-0.01% of the extraction solvent can be added into the extraction solvent; the extraction temperature is 80-120 ℃; the static extraction time is 4-6 min, and the nitrogen purging time is 30-60 s; the number of static cycles is 2-3.
in the step (3), the preferable internal standard substance is deuterated thiabendazole (thiabendazole NH D6), and the standard working curve comprises 5-7 gradient concentrations.
the liquid chromatography conditions in the step (4) are as follows: a chromatographic column: agilent SB-C18, column length 100mm, inner diameter 3.0mm, filler particle size 1.8 μm; mobile phase A: 0.01% by volume of aqueous formic acid solution; mobile phase B: methanol; gradient elution: 40-90% of mobile phase B in 0-4 min, and 90% of mobile phase B in 5-10 min; flow rate: 0.30 mL/min; the column temperature is 40 ℃; the sample injection amount is 5-10 mu L.
Mass spectrum conditions in the step (4): an ion source: electrospray ionization source (ESI), positive mode detection; the temperature of the drying gas is 300 ℃, and the flow rate is 3 mL/min; the temperature of the sheath gas is 250 ℃, and the flow rate is 11 mL/min; the spray gas pressure was 45 psi; scanning mode: multiple reaction monitoring mode (MRM). The mass spectral parameters of 1-hydroxybenzotriazole and internal standard under the above conditions are shown in Table 2.
TABLE 2 Mass Spectrometry parameters of HOBT and internal standards in LC-MS/MS
a bold face is a quantitative ion
The invention establishes a rapid and efficient extraction method by using an accelerated solvent extraction technology, and effectively carries out quantitative analysis on trace 1-hydroxybenzotriazole in soil and plants by using a triple series quadrupole LC-MS after synchronous purification and solid-phase extraction purification.
Compared with the prior art, the invention has the following beneficial effects:
(1) The extraction solvent uses a mixed organic solvent acidified by formic acid, effectively inhibits ionization of the 1-hydroxybenzotriazole and degradation in the extraction process, and obviously improves the recovery rate of the 1-hydroxybenzotriazole.
(2) Silica gel and anhydrous sodium sulfate are added into the extraction tank as matrix adsorbents, synchronous purification is performed in the extraction process, matrix interference on samples such as soil or sediments is effectively reduced, the extraction liquid can be directly operated on a machine for detection after being concentrated, the steps of purification and enrichment are reduced, the experiment time is shortened, the experiment efficiency is accelerated, and the method is particularly suitable for analysis and detection of large-batch samples.
(3) For samples with high pigment and fat contents such as straws, grains and the like, a solid phase extraction and purification step is added after extraction, and matrix interference is further reduced and the sensitivity of the method is improved after purification by using a Florisil solid phase extraction column.
(4) The method uses an internal standard method for quantification, the internal standard substance deuterated thiabendazole has similar properties with the target compound, the retention time in an instrument is close, the internal standard substance deuterated thiabendazole is added into a sample before the sample is extracted, the error caused by the loss of the target compound in the extraction process is offset, and a better recovery rate is obtained in multiple experiments, so that the method is an ideal internal standard substance.
(4) At present, researchers in China pay little attention to 1-hydroxybenzotriazole in the environment, the technology for analyzing and detecting the 1-hydroxybenzotriazole in soil and plants is in the technical blank, and mainly because the matrix in soil and plant samples is complex, so that the matrix interference is serious, and the sensitivity of a conventional detection method is low, so that the analysis is difficult. The invention provides a set of efficient, rapid and accurate extraction and analysis technology of 1-hydroxybenzotriazole in soil and plants from the aspects of pretreatment and instrument analysis, and fills up the technical blank in China at present. The recovery rate of the 1-hydroxybenzotriazole is 92.6 percent, the standard deviation is 2.4 percent, the matrix effect is 90.7 percent, the detection limit is 0.15ng/g, and the quantification limit is 0.51 ng/g; in the plant sample, the recovery rate of the 1-hydroxybenzotriazole is 89.8%, the standard deviation is 1.6%, the matrix effect is 90.3%, the detection limit is 0.69ng/g, and the quantification limit is 2.31ng/g, so that the method is suitable for analyzing and detecting the 1-hydroxybenzotriazole in soil and plant medium.
Drawings
FIG. 1 is a characteristic ion flow diagram of 1-Hydroxybenzotriazole (HOBT) and an internal standard deuterated thiabendazole (thiabendazole NH D6).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Instruments and reagents used in the examples
The target compound 1-hydroxybenzotriazole (HOBT, 99%) and the internal standard deuterated thiabendazole (thiabendazole NH D6, 99%) were purchased from dr. The methanol and the dichloromethane used in the experiment are both imported chromatographic purities, and the water used is Milli-Q water; the anhydrous sodium sulfate, the silica gel and the quartz sand are domestic analytical purities; glassware used in the experiment is cleaned by tap water, washed by Milli-Q water, dried and baked in a muffle furnace for 3-4 hours at the temperature of more than 300 ℃.
The extraction apparatus was a 34mL stainless steel extraction cell equipped with an accelerated solvent extractor ASE 300(Dionex, Sunnyvale, Calif., USA). The detection instrument is a high performance liquid chromatography-mass spectrometer (LC-MS/MS) Agilent1200RRLC/Agilent G6460A Triple Quadrupole.
Example 1
(1) Sample pretreatment
Soil samples were taken at 100g, freed of larger impurities such as stones, freeze-dried, ground and sieved through a 0.90mm sieve.
(2) Accelerated solvent extraction
Extracting a sample by using an accelerated solvent extractor, wherein the extraction process comprises the following steps: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding 2.0g of silica gel, tapping the extraction tank to flatten the surface of the silica gel, then adding 2.0g of anhydrous sodium sulfate, flattening the surface, adding a 5.0g of soil sample, flattening, dropwise adding 100 mu L of 1mg/L internal standard substance deuterated thiabendazole working solution, adding 5.0g of quartz sand after the internal standard solution solvent is volatilized, finally, putting another piece of filter paper for compacting, screwing the extraction tank, and putting the filter paper into an accelerated solvent extraction instrument for extraction. Extracting with methanol/dichloromethane (volume ratio of 90: 10) under the condition of 100 deg.C, adding 0.01% formic acid, static extracting for 5min for 60s under nitrogen purging, and circulating for 2 times. After extraction, the extract is evaporated to near dryness by rotation, then the volume is determined by 1mL of methanol, the solution is filtered through a 0.22 μm organic phase nylon filter membrane, and the solution is transferred to a sample injection vial for detection on a machine.
(3) Preparation of standard working solutions
Weighing standard substances of 1-hydroxybenzotriazole and an internal standard substance deuterated thiabendazole by using an analytical balance, preparing 100mg/L stock solution by using methanol respectively, diluting the stock solution into 1mg/L working solution, and finally preparing standard working solutions with the concentrations of 1, 5, 10, 20, 50, 100 and 200 mug/L, wherein the concentration of the internal standard substance is 100 mug/L, and establishing a standard working curve by using an internal standard method.
(4) High performance liquid chromatography-mass spectrometer detection
The liquid chromatography conditions were: a chromatographic column: agilent SB-C18, column length 100mm, inner diameter 3.0mm, filler particle size 1.8 μm; mobile phase A: 0.01% formic acid in water (by volume); mobile phase B: methanol; gradient elution: 40-90% of mobile phase B in 0-4 min, and 90% of mobile phase B in 5-10 min; flow rate: 0.30 mL/min; the column temperature is 40 ℃; the sample size was 5. mu.L.
Mass spectrum conditions: an ion source: electrospray ionization source (ESI), positive mode detection; the temperature of the drying gas is 300 ℃, and the flow rate is 3 mL/min; the temperature of the sheath gas is 250 ℃, and the flow rate is 11 mL/min; the spray gas pressure was 45 psi; scanning mode: multiple reaction monitoring mode (MRM).
Under the above conditions, the mass spectrum parameters of 1-hydroxybenzotriazole and the internal standard substance deuterated thiabendazole are shown in Table 2.
and (4) measuring the injection of the standard working solution of each concentration gradient in the step (3) in a high performance liquid chromatography-mass spectrometer, and establishing a standard working curve by using an internal standard method. Injecting the sample solution purified in the step (2) into a high performance liquid chromatography-mass spectrometer for determination under the same conditions, calculating the concentration of the 1-hydroxybenzotriazole in the solution through a standard working curve, and then calculating the content of the 1-hydroxybenzotriazole in the sample according to the mass of the sample.
And (3) qualitative identification: under the same instrument condition, if the chromatographic peak retention time of the 1-hydroxybenzotriazole in the sample is consistent with that of the corresponding target compound in the standard working solution, and the abundance ratio of the qualitative ion pair and the quantitative ion pair is the same as that of the standard solution, the existence of the 1-hydroxybenzotriazole in the sample can be judged.
Quantitative analysis: and (3) establishing a standard working curve according to the result of the standard working solution, wherein the linear range is 0-200 mu g/L, the correlation coefficient of the standard curve is more than 0.999, the concentration of the 1-hydroxybenzotriazole is calculated according to the ratio of the quantitative ion peak areas of the 1-hydroxybenzotriazole and the internal standard substance, and finally the concentration of the 1-hydroxybenzotriazole in the sample is calculated according to the sample amount.
Spiked recovery, reproducibility and matrix effect:
The recovery rate adopts 3 times of parallel standard addition, and the standard addition method comprises the following steps: and (3) adding 100 mu L of 1 mg/L1-hydroxybenzotriazole standard solution into a 5.0g soil sample to enable the concentration of the 1-hydroxybenzotriazole in the sample to be 20ng/g, waiting for 30min, completely volatilizing the solvent, adsorbing the 1-hydroxybenzotriazole in the sample, and extracting and detecting according to the step (2). The actual measured spiked sample concentration was subtracted from the blank sample concentration and compared to the theoretical spiked concentration to obtain the recovery of 1-hydroxybenzotriazole, and the process repeatability is expressed as the standard deviation of 3 parallel spiked results. Matrix effect test method: extracting and concentrating the blank sample to 1mL, taking 100 mu L of sample solution, drying on a nitrogen blowing instrument, adding 100 mu L of standard solution with the concentration of 100 mu g/L, carrying out vortex oscillation, and carrying out machine detection. And subtracting the blank sample concentration from the actually measured standard sample concentration, and comparing the blank sample concentration with the theoretical addition concentration to obtain the matrix effect result of the method. The results of the standard addition recovery rate, the repeatability and the matrix effect of the method are shown in Table 3, and it can be seen that under the condition that the standard addition concentration in the soil is 20ng/g, the average recovery rate of the 1-hydroxybenzotriazole is 92.6%, the standard deviation is 2.4% and the matrix effect is 90.7%, which shows that the method has good recovery rate and repeatability and low matrix interference, and can be used for detecting soil samples.
detection limit and quantification limit:
the concentration that corresponds when sample signal intensity reaches 3 times noise intensity is the detection limit, and the concentration that corresponds when sample signal intensity reaches 10 times noise intensity is the ration limit, and concrete operation is: and calculating the signal-to-noise ratio under the standard concentration by using data processing software, and dividing the standard concentration by the signal-to-noise ratio, wherein 3 times of the result is the detection limit of the method, and 10 times of the result is the quantification limit. The detection limit and the quantification limit result of the method are shown in table 3, and it can be seen that under the condition that the standard concentration of the soil is 20ng/g, the detection limit of 1-hydroxybenzotriazole is 0.15ng/g, and the quantification limit is 0.51ng/g, which indicates that the detection method has high sensitivity and can be used for detecting trace 1-hydroxybenzotriazole in a soil sample.
TABLE 3 method recovery, matrix Effect, detection limits and quantitation limits
aMean ± standard deviation (%) (n ═ 3);bmean ± standard deviation (%) (n ═ 3);c(ng/g)；d(ng/g)
Example 2
(1) sample pretreatment
collecting 50g wheat grain sample, removing obvious impurities, freeze drying, and grinding with plant grinder.
(2) Accelerated solvent extraction
Extracting a sample by using an accelerated solvent extractor, wherein the extraction process comprises the following steps: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding 2.0g of silica gel, tapping the extraction tank to flatten the surface of the silica gel, then adding 3.0g of anhydrous sodium sulfate, flattening the surface, adding 5.0g of a wheat grain sample, flattening, dropwise adding 100 mu L of 1mg/L internal standard substance deuterated thiabendazole working solution, adding 2.0g of quartz sand after the internal standard solution solvent is volatilized, finally, putting another piece of filter paper for compacting, screwing the extraction tank, and putting the extraction tank into an accelerated solvent extraction instrument for extraction. Extracting with methanol/dichloromethane (volume ratio of 80: 20) under the conditions of 100 deg.C, static extraction time of 5min and nitrogen purge time of 60s, adding formic acid 0.01% of the total volume of the extracting solvent, and circulating for 2 times. After extraction, the extract was rotary evaporated to near dryness, and then diluted to volume with 1mL of methanol for further purification.
(3) Solid phase extraction purification
Placing a Florisil solid phase extraction column (1000mg, 6mL) on a solid phase extraction device, pre-washing the column (the Florisil solid phase extraction column) by using 10mL dichloromethane and 10mL methanol in sequence to activate the column and remove part of impurities, immediately adding a sample to be purified when the liquid level of a solvent in the column is close to the surface of a filler, receiving a leachate by using a pear-shaped flask, washing a sample bottle by using 5mL methanol, adding a washing solution into the column, and repeating twice. The pear-shaped flask received all the leachates, rotary evaporated to near dryness, fixed volume with 1mL of methanol, filtered with an organic phase nylon filter membrane, transferred to a sample injection vial, and tested.
(4) preparation of standard working solutions
Weighing standard substances of 1-hydroxybenzotriazole and an internal standard substance deuterated thiabendazole by using an analytical balance, preparing 100mg/L stock solution by using methanol respectively, diluting the stock solution into 1mg/L working solution, and finally preparing standard working solutions with the concentrations of 1, 5, 10, 20, 50, 100 and 200 mug/L, wherein the concentration of the internal standard substance is 100 mug/L, and establishing a standard working curve by using an internal standard method.
(5) High performance liquid chromatography-mass spectrometer detection
The liquid chromatography conditions were: a chromatographic column: agilent SB-C18, column length 100mm, inner diameter 3.0mm, filler particle size 1.8 μm; mobile phase A: 0.01% formic acid in water (by volume); mobile phase B: methanol; gradient elution: 40-90% of mobile phase B in 0-4 min, and 90% of mobile phase B in 5-10 min; flow rate: 0.30 mL/min; the column temperature is 40 ℃; the sample size was 5. mu.L.
mass spectrum conditions: an ion source: electrospray ionization source (ESI), positive mode detection; the temperature of the drying gas is 300 ℃, and the flow rate is 3 mL/min; the temperature of the sheath gas is 250 ℃, and the flow rate is 11 mL/min; the spray gas pressure was 45 psi; scanning mode: multiple reaction monitoring mode (MRM).
Under the above conditions, the mass spectrum parameters of 1-hydroxybenzotriazole and the internal standard substance deuterated thiabendazole are shown in Table 2.
And (4) measuring the injection of the standard working solution of each concentration gradient in the step (4) in a high performance liquid chromatography-mass spectrometer, and establishing a standard working curve by using an internal standard method. Injecting the sample solution purified in the step (3) into a high performance liquid chromatography-mass spectrometer for determination under the same conditions, calculating the concentration of the 1-hydroxybenzotriazole in the solution according to a standard working curve, and then calculating the content of the 1-hydroxybenzotriazole in the sample according to the mass of the sample.
and (3) qualitative identification: under the same instrument condition, if the chromatographic peak retention time of the 1-hydroxybenzotriazole in the sample is consistent with that of the corresponding target compound in the standard working solution, and the abundance ratio of the qualitative ion pair and the quantitative ion pair is the same as that of the standard solution, the existence of the 1-hydroxybenzotriazole in the sample can be judged.
Quantitative analysis: and (3) establishing a standard working curve according to the result of the standard working solution, wherein the linear range is 0-200 mu g/L, the correlation coefficient of the standard curve is more than 0.999, the concentration of the 1-hydroxybenzotriazole is calculated according to the ratio of the quantitative ion peak areas of the 1-hydroxybenzotriazole and the internal standard substance, and finally the concentration of the 1-hydroxybenzotriazole in the sample is calculated according to the sample amount.
Spiked recovery, reproducibility and matrix effect:
The recovery rate adopts 3 times of parallel standard addition, and the standard addition method comprises the following steps: and (3) adding 100 mu L of 1 mg/L1-hydroxybenzotriazole standard solution into a 5.0g wheat grain sample to enable the concentration of the 1-hydroxybenzotriazole in the sample to be 20ng/g, waiting for 30min, completely volatilizing the solvent, adsorbing the 1-hydroxybenzotriazole in the sample, and extracting and detecting according to the step (2). The actual measured spiked sample concentration was subtracted from the blank sample concentration and compared to the theoretical spiked concentration to obtain the recovery of 1-hydroxybenzotriazole, and the process repeatability is expressed as the standard deviation of 3 parallel spiked results. Matrix effect test method: extracting and concentrating the blank sample to 1mL, taking 100 mu L of sample solution, drying on a nitrogen blowing instrument, adding 100 mu L of standard solution with the concentration of 100 mu g/L, carrying out vortex oscillation, and carrying out machine detection. And subtracting the blank sample concentration from the actually measured standard sample concentration, and comparing the blank sample concentration with the theoretical addition concentration to obtain the matrix effect result of the method. The results of the standard recovery rate, the repeatability and the matrix effect of the method are shown in table 4, and it can be seen that under the condition that the standard concentration of the standard in the wheat grains is 20ng/g, the average recovery rate of the 1-hydroxybenzotriazole is 89.8%, the standard deviation is 1.6% and the matrix effect is 90.3%, which shows that the method has good recovery rate and repeatability and low matrix interference, and can be used for detecting the plant sample.
Detection limit and quantification limit:
the concentration that corresponds when sample signal intensity reaches 3 times noise intensity is the detection limit, and the concentration that corresponds when sample signal intensity reaches 10 times noise intensity is the ration limit, and concrete operation is: and calculating the signal-to-noise ratio under the standard concentration by using data processing software, and dividing the standard concentration by the signal-to-noise ratio, wherein 3 times of the result is the detection limit of the method, and 10 times of the result is the quantification limit. The detection limit and the quantification limit result of the method are shown in table 3, and it can be seen that under the condition that the standard concentration of the wheat grains is 20ng/g, the detection limit of 1-hydroxybenzotriazole is 0.69ng/g, and the quantification limit is 2.31ng/g, which indicates that the detection method has high sensitivity and can be used for detecting trace 1-hydroxybenzotriazole in a plant sample.
TABLE 4 methods recovery, matrix Effect, detection limits and quantitation limits
aMean ± standard deviation (%) (n ═ 3);bMean ± standard deviation (%) (n ═ 3);c(ng/g)；d(ng/g)
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A method for detecting 1-hydroxybenzotriazole in soil and plants is characterized by comprising the following steps:
(1) Sample pretreatment
pretreating the collected soil sample or plant sample;
(2) Accelerated solvent extraction
extracting the sample pretreated in the step (1) by using an accelerated solvent extraction instrument to obtain a sample solution to be detected; the adopted internal standard solution is a deuterated thiabendazole solution, and the adopted matrix adsorbent is silica gel and anhydrous sodium sulfate; the adopted extraction solvent is a mixed solvent of methanol and dichloromethane, wherein the volume ratio of the methanol is 80-100%, and formic acid with the total volume of 0.005-0.01% of the extraction solvent is added into the extraction solvent;
(3) Preparation of standard working solutions
Weighing standard substances of the 1-hydroxybenzotriazole and the internal standard substance by using an analytical balance, and respectively preparing stock solutions; diluting the two stock solutions respectively to prepare working solutions, and finally preparing a standard working solution with gradient concentration;
(4) High performance liquid chromatography-mass spectrometer detection
Measuring the standard working solution of each concentration gradient in the step (3) in a high performance liquid chromatography-mass spectrometer, and establishing a standard working curve by using an internal standard method; injecting the sample solution to be detected in the step (2) into a high performance liquid chromatography-mass spectrometer under the same conditions for determination, calculating the concentration of 1-hydroxybenzotriazole in the solution through a standard working curve, and then calculating the content of the 1-hydroxybenzotriazole in the sample according to the mass of the sample; the concentration of the 1-hydroxybenzotriazole in the sample solution is in the linear range of the standard working curve, and if the measured concentration exceeds the linear range of the standard working curve, the sample amount is reduced, and then the extraction and the detection are carried out again;
The liquid chromatography conditions were: a chromatographic column: agilent SB-C18, column length 100mm, inner diameter 3.0mm, filler particle size 1.8 μm; mobile phase A: 0.01% by volume aqueous formic acid solution; mobile phase B: methanol; gradient elution: 40-90% of mobile phase B in 0-4 min, and 90% of mobile phase B in 4-10 min; flow rate: 0.30 mL/min; the column temperature is 40 ℃; the sample injection amount is 5-10 mu L;
Mass spectrum conditions: an ion source: electrospray ionization source, positive mode detection; the temperature of the drying gas is 300 ℃, and the flow rate is 3 mL/min; the temperature of the sheath gas is 250 ℃, and the flow rate is 11 mL/min; the spray gas pressure was 45 psi; scanning mode: multiple reaction monitoring mode.
2. The method for detecting 1-hydroxybenzotriazole in soil and plants according to claim 1, wherein in the step (1), the soil sample pretreatment process comprises: after collecting a soil sample, removing impurities, drying, grinding and sieving for later use; the pretreatment process of the plant sample comprises the following steps: and (3) after collecting the plant sample, removing silt, drying, and uniformly crushing by using a plant crusher for later use.
3. the method for detecting 1-hydroxybenzotriazole in soil and plants as claimed in claim 2, wherein the drying is selected from natural air drying or freeze drying, and soil samples are ground through 0.90mm sieve after drying.
4. The method for detecting 1-hydroxybenzotriazole in soil and plants according to claim 1, wherein in the step (2),
When the sample is a soil sample or a plant sample with low pigment content, the extraction process comprises the following steps: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding a certain amount of matrix adsorbent, leveling the surface, adding a pretreated sample, leveling, dropwise adding an internal standard solution, adding quartz sand after the internal standard solution solvent is volatilized, finally, pressing the extraction tank by putting another piece of filter paper, screwing down the extraction tank, and putting the extraction tank into an accelerated solvent extraction instrument for extraction; after extraction, the extract is subjected to rotary evaporation to be nearly dry, and then 1mL of methanol is used for constant volume; transferring the sample to a sample injection vial by using an organic phase nylon filter membrane to be detected;
when the sample is a sample with high pigment or fat content, the sample is purified and then detected on a machine, and the extraction process comprises the following steps: spreading a piece of filter paper at the bottom of a stainless steel extraction tank, adding a certain amount of matrix adsorbent, leveling the surface, adding a pretreated sample, leveling, dropwise adding an internal standard solution, adding quartz sand after the internal standard solution solvent is volatilized, finally, pressing the extraction tank by putting another piece of filter paper, screwing down the extraction tank, and putting the extraction tank into an accelerated solvent extraction instrument for extraction; after extraction, the extract is subjected to rotary evaporation to be nearly dry, and then 1mL of methanol is used for constant volume; taking a Florisil solid-phase extraction column, pre-rinsing the Florisil solid-phase extraction column by using 10mL of dichloromethane and 10mL of methanol in sequence, immediately adding a sample to be purified when the liquid level of a solvent in the column drops to be close to the surface of a filler, receiving a leaching solution by using a pear-shaped flask, rinsing a sample bottle by using 5mL of methanol, adding a rinsing solution into the Florisil solid-phase extraction column, and repeating twice; the pear-shaped flask received all the leachates, rotary evaporated to near dryness, fixed volume with 1mL of methanol, filtered with an organic phase nylon filter membrane, transferred to a sample injection vial, and tested.
5. The method for detecting 1-hydroxybenzotriazole in soil and plants according to claim 4, wherein the sample amount is 2-8 g; the quartz sand is added as a filling agent, and the adding amount is 2-5 g; adding the matrix adsorbent, namely adding 1-3 g of silica gel, flattening the surface, adding 1-3 g of anhydrous sodium sulfate, and properly increasing or decreasing the using amount of the matrix adsorbent according to the sample amount; the silica gel is 80-100 meshes.
6. The method for detecting 1-hydroxybenzotriazole in soil and plants according to claim 4, wherein the extraction conditions are as follows: the capacity of the extraction tank is 34mL, and the extraction temperature is 80-120 ℃; the static extraction time is 4-6 min, and the nitrogen purging time is 30-60 s; the number of static cycles is 2-3.
7. The method for detecting 1-hydroxybenzotriazole in soil and plants according to claim 1, wherein in the step (3), the standard working solution comprises 5 to 7 gradient concentrations.
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